Seminal fluid contains some of the fastest evolving proteins currently known. These seminal fluid proteins (Sfps) play crucial roles in reproduction, such as supporting sperm function, and ...particularly in insects, modifying female physiology and behavior. Identification of Sfps in small animals is challenging, and often relies on samples taken from the female reproductive tract after mating. A key pitfall of this method is that it might miss Sfps that are of low abundance because of dilution in the female-derived sample or rapid processing in females. Here we present a new and complementary method, which provides added sensitivity to Sfp identification. We applied label-free quantitative proteomics to Drosophila melanogaster, male reproductive tissue - where Sfps are unprocessed, and highly abundant - and quantified Sfps before and immediately after mating, to infer those transferred during copulation. We also analyzed female reproductive tracts immediately before and after copulation to confirm the presence and abundance of known and candidate Sfps, where possible. Results were cross-referenced with transcriptomic and sequence databases to improve confidence in Sfp detection. Our data were consistent with 125 previously reported Sfps. We found nine high-confidence novel candidate Sfps, which were both depleted in mated versus, unmated males and identified within the reproductive tract of mated but not virgin females. We also identified 42 more candidates that are likely Sfps based on their abundance, known expression and predicted characteristics, and revealed that four proteins previously identified as Sfps are at best minor contributors to the ejaculate. The estimated copy numbers for our candidate Sfps were lower than for previously identified Sfps, supporting the idea that our technique provides a deeper analysis of the Sfp proteome than previous studies. Our results demonstrate a novel, high-sensitivity approach to the analysis of seminal fluid proteomes, whose application will further our understanding of reproductive biology.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The critical role of Major Histocompatibility Complex (Mhc) genes in disease resistance and their highly polymorphic nature make them exceptional candidates for studies investigating genetic effects ...on survival, mate choice and conservation. Species that harbor many Mhc loci and high allelic diversity are particularly intriguing as they are potentially under strong selection and studies of such species provide valuable information as to the mechanisms maintaining Mhc diversity. However comprehensive genotyping of complex multilocus systems has been a major challenge to date with the result that little is known about the consequences of this complexity in terms of fitness effects and disease resistance.
In this study, we genotyped the Mhc class I exon 3 of the great tit (Parus major) from two nest-box breeding populations near Oxford, UK that have been monitored for decades. Characterization of Mhc class I exon 3 was adopted and bidirectional sequencing was carried using the 454 sequencing platform. Full analysis of sequences through a stepwise variant validation procedure allowed reliable typing of more than 800 great tits based on 214,357 reads; from duplicates we estimated the repeatability of typing as 0.94. A total of 862 alleles were detected, and the presence of at least 16 functional loci was shown - the highest number characterized in a wild bird species. Finally, the functional alleles were grouped into 17 supertypes based on their antigen binding affinities.
We found extreme complexity at the Mhc class I of the great tit both in terms of allelic diversity and gene number. The presence of many functional loci was shown, together with a pseudogene family and putatively non-functional alleles; there was clear evidence that functional alleles were under strong balancing selection. This study is the first step towards an in-depth analysis of this gene complex in this species, which will help understanding how parasite-mediated and sexual selection shape and maintain host genetic variation in nature. We believe that study systems like ours can make important contributions to the field of evolutionary biology and emphasize the necessity of integrating long-term field-based studies with detailed genetic analysis to unravel complex evolutionary processes.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Males compete over mating and fertilization, and often harm females in the process. Inclusive fitness theory predicts that increasing relatedness within groups of males may relax competition and ...discourage male harm of females as males gain indirect benefits. Recent studies in Drosophila melanogaster are consistent with these predictions, and have found that within-group male relatedness increases female fitness, though others have found no effects. Importantly, these studies did not fully disentangle male genetic relatedness from larval familiarity, so the extent to which modulation of harm to females is explained by male familiarity remains unclear. Here we performed a fully factorial design, isolating the effects of male relatedness and larval familiarity on female harm. While we found no differences in male courtship or aggression, there was a significant interaction between male genetic relatedness and familiarity on female reproduction and survival. Relatedness among males increased female lifespan, reproductive lifespan and overall reproductive success, but only when males were familiar. By showing that both male relatedness and larval familiarity are required to modulate female harm, these findings reconcile previous studies, shedding light on the potential role of indirect fitness effects on sexual conflict and the mechanisms underpinning kin recognition in fly populations.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Male reproductive traits such as ejaculate size and quality, are expected to decline with advancing age due to senescence. It is however unclear whether this expectation is upheld across taxa. We ...perform a meta-analysis on 379 studies, to quantify the effects of advancing male age on ejaculate traits across 157 species of non-human animals. Contrary to predictions, we find no consistent pattern of age-dependent changes in ejaculate traits. This result partly reflects methodological limitations, such as studies sampling a low proportion of adult lifespan, or the inability of meta-analytical approaches to document non-linear ageing trajectories of ejaculate traits; which could potentially lead to an underestimation of senescence. Yet, we find taxon-specific differences in patterns of ejaculate senescence. For instance, older males produce less motile and slower sperm in ray-finned fishes, but larger ejaculates in insects, compared to younger males. Notably, lab rodents show senescence in most ejaculate traits measured. Our study challenges the notion of universal reproductive senescence, highlighting the need for controlled methodologies and a more nuanced understanding of reproductive senescence, cognisant of taxon-specific biology, experimental design, selection pressures, and life-history.
Avian malaria (Plasmodium sp.) is globally widespread, but considerable variation exists in infection (presence/absence) patterns at small spatial scales. This variation can be driven by variation in ...ecology, demography, and phenotypic characters, in particular those that influence the host's resistance. Generation of reactive oxygen species (ROS) is one of the host's initial immune responses to combat parasitic invasion. However, long-term ROS exposure can harm the host and the redox response therefore needs to be adjusted according to infection stage and host phenotype. Here we use experimental and correlational approaches to assess the relative importance of host density, habitat composition, individual level variation and redox physiology for Plasmodium infection in a wild population of great tits, Parus major.
We found that 36% of the great tit population was infected with Plasmodium (22% P. relictum and 15% P. circumflexum prevalence) and that patterns of infection were Plasmodium species-specific. First, the infection of P. circumflexum was significantly higher in areas with experimental increased host density, whereas variation in P. relictum infection was mainly attributed to age, sex and reproduction. Second, great tit antioxidant responses - total and oxidizied glutathione - showed age- , sex- and Plasmodium species-specific patterns between infected and uninfected individuals, but reactive oxygen metabolites (ROM) showed only a weak explanatory power for patterns of P. relictum infection. Instead ROM significantly increased with Plasmodium parasitaemia.
These results identify some key factors that influence Plasmodium infection in wild birds, and provide a potential explanation for the underlying physiological basis of recently documented negative effects of chronic avian malaria on survival and reproductive success.
The ability of female insects to retain and use sperm for days, months, or even years after mating requires specialized storage organs in the reproductive tract. In most orders, these organs include ...a pair of sclerotized capsules known as spermathecae. Here, we report that some
females exhibit previously uncharacterized structures within the distal portion of the muscular duct that links a spermatheca to the uterus. We find that these 'spermathecal duct presences' (SDPs) may form in either or both ducts and can extend from the duct into the sperm-storing capsule itself. We further find that the incidence of SDPs varies significantly between genotypes, but does not change significantly with the age or mating status of females, the latter indicating that SDPs are not composed of or stimulated by sperm or male seminal proteins. We show that SDPs affect neither the number of first male sperm held in a spermatheca nor the number of offspring produced after a single mating. However, we find evidence that SDPs are associated with a lack of second male sperm in the spermathecae after females remate. This raises the possibility that SDPs provide a mechanism for variation in sperm competition outcome among females.
Reproductive ageing can occur due to the deterioration of both the soma and germline. In males, it has mostly been studied with respect to age-related changes in sperm. However, the somatic component ...of the ejaculate, seminal fluid, is also essential for maintaining reproductive function. Whilst we know that seminal fluid proteins (SFPs) are required for male reproductive success across diverse taxa, age-related changes in SFP quantity and composition are little understood. Additionally, only few studies have explored the reproductive ageing of the tissues that produce SFPs, and the resulting reproductive outcomes. Here we provide a systematic review of studies addressing how advancing male age affects the production and properties of seminal fluid, in particular SFPs and oxidative stress, highlighting many open questions and generating new hypotheses for further research. We additionally discuss how declines in function of different components of seminal fluid, such as SFPs and antioxidants, could contribute to age-related loss of reproductive ability. Overall, we find evidence that ageing results in increased oxidative stress in seminal fluid and a decrease in the abundance of various SFPs. These results suggest that seminal fluid contributes towards important age-related changes influencing male reproduction. Thus, it is essential to study this mostly ignored component of the ejaculate to understand male reproductive ageing, and its consequences for sexual selection and paternal age effects on offspring.
Abstract Advancing male age is often hypothesized to reduce both male fertility and offspring quality due to reproductive senescence. However, the effects of advancing male age on reproductive output ...and offspring quality are not always deleterious. For example, older fathers might buffer the effects of reproductive senescence by terminally investing in reproduction. Similarly, males that survive to reproduce at an old age might carry alleles that confer high viability (viability selection), which are then inherited by offspring, or might have high reproductive potential (selective disappearance). Differentiating these mechanisms requires an integrated experimental study of paternal survival and reproductive performance, as well as offspring quality, which is currently lacking. Using a cross-sectional study in Drosophila melanogaster, we test the effects of paternal age at conception (PAC) on paternal survival and reproductive success, and on the lifespans of sons. We discover that mating at an old age is linked with decreased future male survival, suggesting that mating-induced mortality is possibly due to old fathers being frail. We find no evidence for terminal investment and show that reproductive senescence in fathers does not onset until their late-adult life. Additionally, we find that as a father’s lifespan increases, his probability of siring offspring increases for older PAC treatments only. Lastly, we show that sons born to older fathers live longer than those born to younger fathers due to viability selection. Collectively, our results suggest that advancing paternal age is not necessarily associated with deleterious effects for offspring and may even lead to older fathers producing longer-lived offspring.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Spatially-variable processes can be an important element of host–parasite interactions, but their longer term demographic and evolutionary effects depend on the magnitude of variation in space, the ...scale at which variation occurs and the degree to which such processes are temporally stable. Here, we use multiple years of data from a study of two closely related tit species (Paridae), infected with two congeneric species of avian malaria (Plasmodium), to evaluate the roles of extrinsic and intrinsic factors in driving spatial heterogeneity in infection risk, and to address questions of scale and temporal stability in these vector-driven host – parasite interactions. We show that the two malaria parasite species exhibit markedly different spatial epidemiology: P. relictum infections are effectively randomly distributed in space, with no temporal consistency, whereas P. circumfiexum infections exhibit pronounced spatial structuring that is stable over the six years of this study and similar in both host species. We show that both conspecific and heterospecific host density contribute to elevated infection risk, but that the main determinants of elevated risk of P. circumfiexum infection risk are habitat features probably associated with vector distribution and abundance. We discuss the implications of these findings, both for our understanding of the epidemiology of malaria in the wild, but also in terms of the longer-term evolutionary and demographic consequences that spatially variable parasite-mediated selection may have on host populations.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NMLJ, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
A distinguishing characteristic of genes that code for the major histocompatibility complex (MHC) is that alleles often share more similarity between, rather than within species. There are two likely ...mechanisms that can explain this pattern: convergent evolution and trans-species polymorphism (TSP), in which ancient allelic lineages are maintained by balancing selection and retained by descendant species. Distinguishing between these two mechanisms has major implications in how we view adaptation of immune genes. In this study we analyzed exon 2 of the MHC class IIB in three passerine bird species in the genus Corvus: jungle crows (Corvus macrorhynchos japonensis) American crows (C. brachyrhynchos) and carrion crows (C. corone orientalis). Carrion crows and American crows are recently diverged, but allopatric, sister species, whereas carrion crows and jungle crows are more distantly related but sympatric species, and possibly share pathogens linked to MHC IIB polymorphisms. These patterns of evolutionary divergence and current geographic ranges enabled us to test for trans-species polymorphism and convergent evolution of the MHC IIB in crows. Phylogenetic reconstructions of MHC IIB sequences revealed several well supported interspecific clusters containing all three species, and there was no biased clustering of variants among the sympatric carrion crows and jungle crows. The topologies of phylogenetic trees constructed from putatively selected sites were remarkably different than those constructed from putatively neutral sites. In addition, trees constructed using non-synonymous substitutions from a continuous fragment of exon 2 had more, and generally more inclusive, supported interspecific MHC IIB variant clusters than those constructed from the same fragment using synonymous substitutions. These phylogenetic patterns suggest that recombination, especially gene conversion, has partially erased the signal of allelic ancestry in these species. While clustering of positively selected amino acids by supertyping revealed a single supertype shared by only jungle and carrion crows, a pattern consistent with convergence, the overall phylogenetic patterns we observed suggest that TSP, rather than convergence, explains the interspecific allelic similarity of MHC IIB genes in these species of crows.
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